Substituted benzyl carbazic acid esters

ABSTRACT

Compositions and method of treating rhinoviral infections employing substituted benzylidene carbazic acid esters are described.

United States Patent [191 Dusza et al.

1451 Feb. 18, 1975 SUBSTITUTED BENZYL CARBAZIC ACID ESTERS [73] Assignee: American Cyanamid Company,

Stamford, Conn.

22 Filed: Jan. 9, 1974 21 Appl. No.: 432,103

[52] US. Cl. 260/471 C, 424/300 [51] Int. Cl. C07c 125/06 [58] Field of Search 260/471 C [56] References Cited UNITED STATES PATENTS 3,770,695 ll/l973 Nyquist et a] 260/47l C Primary ExaminerLorraine A. Weinberger Assistant ExaminerL. A. Thaxton Attorney, Agent, or FirmErnest Y. Miller [57] ABSTRACT Compositions and method of treating rhinoviral infections employing substituted benzylidene carbazic acid esters are described.

2 Claims, N0 Drawings 1 SUBSTITUTED BENZYL CARBAZIC ACID ESTERS PRIOR ART The following prior art is made of record in connection with this application; l Rabjohn, N. and Barnstroff, H. D. Carbazic Acid Esters as Carbonyl Reagents, (2) Journal of the American Chemical Society, 75, 2259-2261 (1953) and references cited therein, (3) Chemical Abstracts, Vol. 59, p. 2715 (1963), (4) Abstracts Imperial Chemical Industries, Ltd. France Pat. No. l,3l4,362 (Jan. l l, 1963), Related Brit. Appl. Dec. 23, 1958,]une 22 and Nov. 1 1, I959. These references do not disclose the present compounds as useful in rhinoviral infections.

DESCRIPTION OF THE INVENTION This invention relates to methods for the prevention and treatment of rhinoviral infections. In particular, this invention consists of methods of inhibition of the growth of the common cold viruses (rhinovirus) with pharmaceutical compositions containing a substituted ethylidene carbazic acid ester and a pharmaceutical carrier.

The substituted benzylidene carbazic acid esters which are useful in the treatment of rhinoviral infections may be described by the following formula:

R N-NHCOO-alkyl wherein R is hydrogen, phenyl or lower alkoxy (CFCG), R1 and R are each hydrogen, chloride, lower alkyl (C -C or lower alkoxy (C -C R is hydrogen or hydroxy, R is hydrogen or lower alkyl (C -C and alkyl radical is one consisting ofC to C carbon atoms, with the proviso that when R and R are both methoxy and R and R are both hydrogen, then R, and alkyl are both ethyl.

The active components ofthis invention are prepared according to the following method.

wherein R, R R R and R and alkyl are defined as above.

Upon completion of the reaction, the mixture is evaporated under reduced pressure and the residue is 5 treated with water, the resultant precipitate filtered and recrystallized from an appropriate solvent to provide the desired carbazate ester.

Among the specific compounds which can be prepared by the above method are, for example: 3-lpchlorobenzylidene)-carbazic acid ethyl ester, 3-(3,4,5- trimethoxybenzylidene)-carbazic acid ethyl ester, 3-(pchloro-a-methylbenzylidene)carbazic acid ethyl ester, 3-(a-ethylveratrylidene)-carbazic acid ethyl ester, 3- (3,4-dichloro-a-methylbenzylidene )-carbazic acid ethyl ester, 3-(3-ethoxysalicylidene)-carbazic acid ethyl ester, 3-(3,4-dichlorobenzylidene)-carbazic acid ethyl ester, 3-(3,4-dichloro-a-ethylbenzylidene)- carbazic acid ethyl ester, 3-(m-a-dimethylbenzylidene)-carbazic acid ethyl ester, 3-(p,a-

dimethylbenzylidene)-carbazic acid ethyl ester, 3- (a,3,4-trimethylbenzylidene)-carbazic acid ethyl ester, 3-l1-(3-biphenylyl)ethylidene]-carbazic acid ethyl ester, 3-(3-methoxy-a-methylbenzylidene)-carbazic acid ethyl ester, 3-(3,5-dimethoxy-a-methylbenzylidene)- carbazic acid ethyl ester, 3-(3-ethoxysalicylidene)- carbazic acid methyl ester, 3-[ l-(3-biphenylyl)ethylidene1-carbazic acid prolyl ester and 3-(3-ethoxysalicylidene)-carbazic acid butyl ester.

The active components of this invention may be formulated into compositions for use as antirhinoviral agents by methods well known to the skilled pharmaceutical chemist. When compositions were intended to be administered orally, preferably in the form of a tablet or capsule, they can be prepared in the usual manner or formulated into a sustained release preparation by methods well known in the art.

A preferred mode of administration for effecting antirhinoviral activity is by intranasal application, preferably in the form of a suspension or solution which is sprayed into the nasal cavity and having a concentration of from mg. to 300 mg. per cubic centimeter.

A furtherform of administration can be by intramuscular or subcutaneous injection.

The tablets and capsules are prepared by known methods and may include the usual pharmaceutical excipients such as sucrose, starch, lactose, magnesium stearate, etc. These oral compositions are administered in 2 to 4 doses of 50 mg. to 400 mg. per dose totaling about lO0 to 1,600 mg. per day. The intranasal formulation is best administered as a 0.5-l0% suspension in the form of a spray or nose drops several times a day. The injectable formulation can be administered once daily at a concentration of 0.5 to 2.0 mg./kg. of body weight.

The compositions are preferably administered to a warm-blooded animal prior to rhinoviral infection in order to prevent or ameliorate the infection, soon after known exposure to infection, or upon recognition of symptoms in order to treat the infection and minimize its systemic effects.

A representative formulation for a tablet containing, for example, the antirhinoviral compound 3-(aethylveratrylidene)-carbazic acid ethyl ester is:

Active compound 250 mg. Calcium carbonate mg. Sucrose 88 mg. Starch 20 mg. Magnesium stearatc 5 mg.

A representative formula for a capsule containing the above antirhinoviral compound is:

for this purpose was of the following formulation:

Minimum Essential Medium (Eagles) containing Earle's Salts (Grand Island Biological Company. Grand Island, N. Y.) and to which has been added Active compound 250 mg. 5 lonagm. 2 049/ Lactose. 50 Diethylaminoethyl dextran 0.01 Milgnfilum Stea -ate 7 (I tg H Magnesium chloride 0.069:

V V lfetal call: serum 297 (\'/i') The virus-infected cell monolayers plus test com- A representative formulation for an intranasal spray 10 pound were incubated for 3 to 5 daysv in a humidified suspension containing the above antirhinoviral comatmosphere of 5% carbon dioxide at either 33 or pound is: 37C., depending on the virus. The ability of these compounds to protect tissues from destruction by the viruses was then evident after staining the residual, unin- Aciive compound l5 fected, cells with 0.5% crystal violet in ethanol. gg 'g w/V A summary of the test results obtained with represen- Sodium citrate 02% w/v tative compounds is shown in Table I.

TABLE I Rhinoviruses Compound 18 2 5 14 23 3-(p-chlorobenzylidene)-carbazic acid ethyl ester 3-(3,4,S-trimethoxybenzylidene)-carbazic acid ethyl ester 3-(p-chloro-a-methylbenzylidene)-carbazic acid ethyl ester 3-(3,4-dichloro-a-methylbenzylidene)- carbazic acid ethyl ester 3-ethoxysalicylidene carbazic acid ethyl ester 3-(3,4-dichlorobenzylidene)-carbazic acid ethyl ester 3-(3,4-dichloro-a-ethylbenzylidene)- carbazic acid ethyl ester V 3 -(m,u-dimethylbenzylidenefcarbazic acid ethvl ester 3-(p.g;dimethylbenzylidene) carbazic acid ethyl ester M 3-(a,3,4-trimethylbenzylidene)- carbazic acid ethyl ester 3-[ l-(S-biphenylyl)ethylidenelcarbazic acid ethyl ester Protectsiissue from destruction by'virus.

Potassium biphlhalate 0.13% w/v In addition to the above, the following three com- Eucalyptol 0.02% w/v Trimerosal 000W w/v pounds were also tested by the above procedure: Purified water qs to 100% w/v N-NHC00C H The compounds of the present invention exhibit'antiviral activity against a variety of rhinoviruses The folc330 C C H lowing tests were carried out using the active compo- 2 5 nents of this invention. Confluent monolayers of a continuous gellline such as HeLa, H Ep -2 KB Oil 1 32 2 C11 0 B-(a-ethylveratrylidene) -carbazic acid ethyl es ter grown in plastic tissue culture dishes were infected with one of the viruses causing respiratory illness such as the common cold.These viruses include members of the picornaviruses group including the rhinoviruses, for example, types lB, 2, 5, 14 or 23 and including the env ry esliqaexemijstC reesgsAr1i9%:21319:

tection of the tissues to the cytopathic effects of the viruses was ascertained by means of a plaque inhibition test in which the test compound was adsorbed into a til- C CH O H Gila 3- (3 tdimethoxybenzy1idene -carbaz1c acid ethyl N-NHCOOCgHg C cs o ter paper disc and placed on the agar used to overlay the infected cell monolayers, or by incorporation into the said agar overlay. The agar overlay medium used ethyl ester The results are summarized in Table 11. 5 10.4 gm. ofethyl carbazate and 1 drop ofconcentrated TABLE 11 Rhinoviruses Compound 18 2 5 14 23 3 (a-ethylveratrylidene)-earbazic acid ethyl ester 3'( 3,4-dimethoxybenzylidene )-carbazic acid ethyl ester 3(3,4-dimethoxy-a-methylbenzylidene)- carbazic acid ethyl ester Protects tissue from destruction by virus.

In addition 3-(a-ethylveratrylidene)-carbazic acid ethyl ester is also active in providing protection against coxsackie A-21 virus.

The results in Table 11 are separately presented to point out the specificity of antirhinoviral activity as re lated to chemical structure. It can be seen that 3-(ozethylveratrylindene)-carbazic acid ethyl ester is an extremely active antirhinviral agent. while the two closely related homologs are inactive.

SPECIFIC DISCLOSURE The following examples describe the preparation of representative compounds of the invention.

EXAMPLE 1 Preparation of 3-(p-Chlorobenzylidene)-carbazic acid ethyl ester A mixture of 9.8 gm. of 3,4,5-trimethoxybenzaldehyde, 5.2 gm. of ethyl carbazate and 0.5 ml. of glacial acetic acid in 50 ml of ethanol is refluxed overnight and then cooled. The solid which forms is collected by filtration, washed with ethanol and dried yielding 6.2 gm., melting point l28l30C. Analysis calculated for: C, H,,,N- O C, 55.31; H, 6.43; N, 9.92. Found: C, 55.84; H, 6.50; N, 10.34.

EXAMPLE 3 Preparation of 3-(p-Chloro-a-methylbenzylidenel-carbazic acid ethyl ester A mixture of 15.46 gm. of 4-chloroacetophenone,

hydrochloric acid in 50 ml. of ethanol is warmed on a steam bath for 15 minutes and then allowed to stand at room temperature overnight. The crystals which form are recovered by filtration, washed with isopropanol A mixture of 5.0 gm. of 3,4-tlimcthoxypropiophcnone, 2.71 gm of ethyl carbazate and 2 drops of concentrated hydrochloric acid in ml. of 9591' ethanol is refluxed on a steam bath for 1 hour. The mixture is cooled overnight and then evaporated to dryness yielding an oil. This oil is triturated with hot hexane yielding a crystalline product. This product is dissolved in methylene chloride and passed through a column of hydrous acid silicate of magnesium. To the refluxing effluent is added hexane until crystallization. The product is collected by filtration and dried. Analysis calculated for: C H N O' C, 59.48; H, 7.19; N, 9.99. Found: C, 59.70; H, 7.52; N, 10.00.

EXAMPLE 5 Preparation of 3-(3,4-Dichloro-a-methylbenzylidene)-carbazic acid, ethyl ester A mixture of 3.78 gm. of 3,4-dichloroacetophen0ne, 2.08 gm of ethyl carbazate and 2 drops of concentrated hydrochloric acid in 100 ml'of 95% ethanol is refluxed for 1 hour on a steam bath and then evaporated to dryness. The product is recrystallized twice from a mixture of acetone and hexane, yielding 1.63 gm, melting point l32133C. Analysis calculated for: cuH gclgNgogi C, 48.02; H, 4.40; N, 10.19; Cl, 25.77. Found: C, 47.69; H, 4.31; N, 10.28; Cl 26.10.

EXAMPLE 6 Preparation of 3-(3-Ethoxysalicylidene)-carbazic acid, ethyl ester A mixture of 4.98 gm. of 3-ethoxysalicylaldehyde, 3.12 gm. of ethyl carbazate and 2 drops of concentrated hydrochloric acid in ml. of 95% ethanol is heated on a steam bath for 1 hour and then evaporated to dryness. The product is recrystallized twice from acetone-hexane, yielding 5.75 gm., melting point l43144C. Analysis calculated for: C H N O C, 57.13; H, 6.39; N, 11.11. Found: C, 57.25; H, 6.34; N, 10.97.

EXAMPLE 7 Preparation of 3-(3,4-Dichlorobenzylidene)-carbazic acid, ethyl ester A mixture of 17.5 gm. of 3,4-dichlorobenzaldehyde and 10.4 gm. of ethyl carbazate in 50 ml. of absolute ethanol containing 2 drops of concentrated hydrochloric acid is refluxed for 1 hour and then cooled. The precipitate is filtered and washed with ethanol. Water is added to the filtrate causing reprecipitation ofthe ethanol soluble product. The two precipitates are combined, dissolved in methylene chloride and passed through a column of acid silicate of magnesium. To the refluxing effluent is added hexane until crystallization. The product is collected, washed with hexane and dried, yielding 9.9 gm.. melting point 132133.5C. Analysis calculated for: C H CI N- O C, 46.00; H, 3.86; Cl, 27.16; N, 10.73. Found: C, 46.28; H, 3.86; Cl, 27.76; N, 10.84.

EXAMPLE 8 Preparation of 3-(3.4-Dichloro-a-ethylbenzylidene)-carbazic acid. ethyl ester A mixture of 4.0 gm. of 3.4-dichloropropiophenone and 2.1 gm of ethyl carbazate in ml. ofabsolute ethanol containing 2 drops of concentrated hydrochloric acid is refluxed for 1.5 hours. The precipitate is collected by filtration. dissolved in methylene chloride and passed through an acid silicate of magnesium column. To the refluxing effluent is added hexane to crystallization. The product is collected by filtration and dried, yielding 4.4 gm., melting point l-153.5C. Analysis calculated for: C H Cl N O- z C, 49.84; H, 4.88; Cl, 24.52; N. 9.69. Found: C, 49.63; H, 4.72; C1, 24.67; N, 9.66.

EXAMPLE 9 Preparation of 3-(m,o:-Dimethylben2ylidene)-carbazic acid, ethyl ester A mixture of 13.42 gm. of m-methylacetophenone and 10.4 gm. of ethyl carbazate in 50 ml. of absolute ethanol containing 2 drops of concentrated hydrochloric acid is refluxed for 1 hour and then cooled. One half of the ethanol is removed and water is added. The precipitate is collected, dissolved in methylene chloride and passed through an acid silicate of magnesium column. To the refluxing effluent is added hexane to crystallization. The product is collected by filtration and dried, yielding 19.5 gm., melting point 77-80C. Analysis calculated for: C H N O C, 65.43; H, 7.32; N, 12.72. Found: C. 65.53; H, 7.28; N, 12.77.

EXAMPLE 10 Preparation of 3-(p.a-Dimethylbenzylidene)-carbazic acid, ethyl ester A mixture of 13.42 gm. of p-methylacetophenone and 10.4 gm. of ethyl carbazate in 50 ml. of absolute ethanol containing 2 drops of concentrated hydrochloric acid is refluxed for 1 hour and then cooled to room temperature. The precipitate is collected by filtration. washed successively with ethanol and water to give 16.6 g. of the carbazate ester, melting point l081 1 1C. The precipitate is dissolved in methylene chloride and passed through an acid silicate of magnesium column. To the refluxing effluent is added hexane to crystallization. The product is collected by filtration and dried yielding 15.8 gm.. melting point 113-1l5C. Analysis calculated for: C,- H,.,N O C. 65.43; H. 7.32; N. 12.72. Found: C6583; H, 7.50: N. 12.84.

EXAMPLE 11 Preparation of 3-((1,3,4-Trimethylbenzylidene)-carbazic acid. ethyl ester A mixture of 24.82 gm. of 3,4'- dimethylacetophenone and 104 gm. of ethyl carbazate in 50 ml. of absolute ethanol containing 2 drops ofconcentrated hydrochloric acid is refluxed for 2 hours and then cooled. The product is collected by filtration. dissolved in methylene chloride and passed through an acid silicate of magnesium column. To the refluxing effluent is added hexane to crystallization. The product is collected by filtration and dried. yielding 19.2 gm.. melting point 105-106.5C. Analysis calculated for: C H N O C, 66.64; H, 7.74; N, 11.96. Found: C. 66.94; H, 7.86; N, 12.04.

EXAMPLE 12 Preparation of 3-[1-(3-Biphenylyl)ethy1idene]-carbazic acid, ethyl ester A mixture of 2.5 gm. of 3-acetylibphenyl and 2.5 gm. of ethyl carbazate in 10 m1. of absolute ethanol containing 2 drops of concentrated hydrochloric acid is heated on a steam bath for 3 hours. Water is added and gum forms. This gum is dissolved in methylene chloride and passed through an acid silicate of magnesium column. The refluxing effluent is treated with hexane to crystallization. The methylene chloride-acid silicate of magnesium-hexane treatment is repeated. The product is collected by filtration, recrystallized from acetonehexane and dried, yielding 1.30 gm., melting point 107107.5C.

We claim:

1. The compound 3-(a-ethylveratrylidenel-carbazic acid ethyl ester.

2. The compound 3-(3-ethoxysalicylidene)-carbazic 

1. THE COMPOUND 3-(A-ETHYLVERATRYLINDENE)-CARBAZIC ACID ETHYL ESTER.
 2. The compound 3-(3-ethoxysalicylidene)-carbazic acid ethyl ester. 